Proteolysis is a key component of many important biochemical and physiological events. Under normal conditions (trophoblast implantation, embryonic morphogenesis, tissue remodelling, angiogenesis) it is limited in duration and strictly regulated. Under pathological conditions, however, these regulatory controls fail. For example, malignant neoplasms are characterized by an imbalance of proteolysis which favors invasion. A critical enzyme in the process of malignant conversion is the metalloproteinase, collagenase. Our laboratory has recently identified, purified, characterized and obtained amino acid sequence of a cartilage- derived inhibitor (CDI) of collagenase. Interestingly, CDI is also a potent inhibitor of angiogenesis in vitro and in vivo. We have recently extended these studies to include chondrosarcoma, the malignant tumor of cartilage. Our preliminary results suggest that in chondrosarcoma, in contrast to normal cartilage, the inherent proteolytic balance appears to be shifted in favor of at least one metalloproteinase activity, collagenase. We are now interested in understanding the role of CDI and its target metalloproteinases in the development of, and the transition, to the angiogenic phenotype during tumorigenesis. Towards this end, we propose the following Specific Aims: 1. To develop a model system to be utilized in the study of the role of CDI, its target metalloproteinases and FGF on the switch to the angiogenic phenotype during tumor development. An in vivo model system will be developed in which three-dimensional nodules representative of each of the presumptive stages in the transition to the angiogenic state (avascular, prevascular, vascular) during the development of chondrosarcoma can be predictably obtained and studied. Differentiated chondrosarcoma chondrocyte cell lines representative of each of these transition stages will be established as will clonal cell lines of each type. 2. To correlate the presence and activity of CDI, its target enzymes and FGF by these chondrosarcoma chondrocytes with the onset and development of the angiogenic phenotype. Radiometric enzyme assays, sodium dodecyl sulfate-substrate gel electrophoresis and 3H-thymidine incorporation assays will be employed to determine the activity and amount of these molecules of interest. The relationship between the levels of these factors and the onset and development of the angiogenic state during tumorigenesis will be characterized. 3. To utilize recombinant DNA technology to modulate the production of CDI in normal and chondrosarcoma chondrocytes. Chondrosarcoma chondrocyte cell lines from avascular and vascular nodules will be transfected with plasmid constructs capable of expressing """"""""antisense"""""""" and """"""""sense"""""""" CDI respectively. The effect of these genetic modifications on the expression of CDI will be determined. 4. To study the phenotypic consequences of genetically altered CDI expression on the switch to the angiogenic state. In vivo studies will be utilized to investigate the angiogenic potential of chondrocytes which were up-regulated and down-regulated for CDI expression. We will also investigate the tumorigenic potential of these genetically modified cells in athymic mice.
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